Biology subjects

Cho, Y.

Publications and source records attributed to Cho, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Asymmetric distribution of mitochondrial Ca2+ regulators specifies compartment-specific mitochondrial function and neuronal development

Neuronal polarization is essential for functional compartmentalization, enabling dendritic synaptic integration and axonal action potential generation. While structural differences in mitochondria across compartments have been identified, their functional distinctions remain unclear. Here, we uncovered compartment-specific mitochondrial Ca2+ dynamics and their molecular determinants. In axonal mitochondria, Ca2+ uptake through MCU occurs independently of ER-stored Ca2+ release, with faster matrix Ca2+ clearance than dendritic mitochondria, where Ca2+ uptake predominantly originates from ER Ca2+. The ER-independent mitochondrial Ca2+ uptake in axonal mitochondria is mediated by enriched MCU-regulating proteins, MICU1 and MICU2, while higher NCLX expression facilitates rapid Ca2+ clearance. Moreover, NCLX knockdown, which functionally mimics a mental retardation-associated mutation, caused more significant axonal branching defects compared to dendrites in vivo, aligning with its enrichment in axons. These findings highlight fundamental Ca2+-modulating features and developmental importance of neuronal mitochondria in a compartment-specific manner and reveal the key underlying molecular mechanisms.

neuroscience↗

bioRxiv2025

Modeling cellular influence delineates functionally relevant cellular neighborhoods in primary and metastatic pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, with liver metastases significantly worsening outcomes. However, distinct features of the tumor microenvironment (TME) between primary and metastatic sites remain poorly defined. Cellular neighborhoods within the TME are recognized as functional units that influence tumor behavior. Conventional spatial methods, which assign equal weights to all cells in a region, fail to capture the nuances of cellular interactions. To address this, we developed Functional Cellular Neighborhood (FunCN) quantification, which integrates both the proportion and proximity of surrounding cells. Applying FunCN to PDAC imaging mass cytometry data, we identified neutrophil-enriched interactions in liver metastases compared to primary tumors, correlating with elevated VISTA expression by tumor cells. Additionally, FunCN clusters around CD8+ T cells in pancreas and liver were associated with higher TIGIT and LAG3, respectively. These findings demonstrate the importance of spatial immune landscapes in PDAC and identify potential therapeutic opportunities.

systems biology↗

bioRxiv2025

Structural mechanism of insulin receptor activation by a dimeric aptamer agonist

Insulin binding to the insulin receptor (IR) triggers signaling pathways that regulate glucose uptake and cell growth. In previous work, we identified a DNA aptamer, A62, which partially activates the IR. During engineering aptamers for improved in vivo stability, we discovered that crosslinking two A62 aptamers with linkers of varying lengths led to full phosphorylation of the IR, though activation remained selective to the AKT pathway. To elucidate the mechanism behind this aptamer-induced full activation of the IR, we determined the structure of the IR in complex with a dimeric form of A62 (A62D) linked by an 8-nucleotide connector. We identified three distinct conformations of the IR: arrowhead-shaped, pseudo-arrowhead-shaped, and pseudo-gamma-shaped. The pseudo-gamma-shaped conformation closely resembles the structure of a fully active IR bound by a single insulin molecule. In these configurations, only one A62 monomer (A62M) within the A62D dimer binds to the IR dimer. This binding brings the IR monomers into close proximity, promoting intermolecular trans-phosphorylation. Our findings provide valuable structural insights for the development of novel therapeutic strategies targeting the IR.

biochemistry↗

bioRxiv2025

BoltzDesign1: Inverting All-Atom Structure Prediction Model for Generalized Biomolecular Binder Design

Deep learning in structure prediction has revolutionized protein research, enabling large-scale screening, novel hypothesis generation, and accelerated experimental design across biological domains. Recent advances, including RoseTTAFold-AA and AlphaFold3, have extended structure prediction models to work with small molecules, nucleic acids, ions, and covalent modifications. We present BoltzDesign1, which inverts the Boltz-1 model, an open source reproduction of AlphaFold3, to enable the design of protein binders for diverse molecular targets without requiring model finetuning. By utilizing only the Pairformer and Confidence modules, our method significantly reduces computational costs while achieving outstanding in silico success rates and diversity in binder generation. Optimizing directly on the distogram allows us to shape the probability distribution of atomic distances, rather than adjusting a single structure, steering the design toward sequences that yield robust structures with well-defined energy minima. By leveraging a fully atomic model trained on a wide variety of macromolecules, we can generate diverse heterocomplexes with flexible ligand conformations--a capability not currently matched by existing methods. This approach enables the design of novel protein interactions with potential applications in biosensors, enzyme engineering, therapeutic development, and biotechnological innovations.

molecular biology↗

bioRxiv2025

Designed Peptides as Affinity Ligands for Extracellular-Vesicle-based Cancer Biomarker Detection

Assays for cancer diagnosis via the analysis of tumor biomarkers on circulating extracellular vesicles (EVs) have shown great potential. Single EV imaging that can measure the abundance of protein biomarkers in EVs can help in detecting the presence, stage, and progression of disease. Antibodies are typically used to detect EV proteins. Controlling the quality of antibody-based immunoassays can, however, be challenging, as they may exhibit unintended cross-reactivity with non-target proteins or variability in binding affinity across different batches, even for monoclonal antibodies. Here, we report short peptides that are a promising alternative to antibodies for detecting protein biomarkers in EVs. We describe an effort that combines a Peptide Binding Design (PepBD) algorithm and molecular-level simulations to identify peptides that can recognize (1) the extracellular domain of EpCAM (a known cancer biomarker) and (2) the extracellular domain of tetraspanin CD81(a protein commonly expressed on the surface of EVs). The peptides designed for EpCAM and CD81 were labeled with a fluorescent dye and their binding to the target protein was evaluated using fluorescence ELISA. The results demonstrated that one of the computationally designed peptides EP-2.1 exhibited an affinity for the target EpCAM protein comparable to that of an antibody. Further testing involved single EV imaging to gauge the peptides affinity towards EVs. Designed peptides for both the target proteins showed similar affinity as antibodies to EVs.

cancer biology↗

bioRxiv2025

Selective manipulation of excitatory and inhibitory neurons in top-down and bottom-up visual pathways using ultrasound stimulation

IntroductionTechniques for precise manipulation of neurons in specific neural pathways are crucial for excitatory/inhibitory (E/I) balance and investigation of complex brain circuits. Low-intensity focused ultrasound stimulation (LIFUS) has emerged as a promising tool for noninvasive deep-brain targeting at high spatial resolution. However, there is a lack of studies that extensively investigate the modulation of top-down and bottom-up corticothalamic circuits via selective manipulation of excitatory and inhibitory neurons. Here, a comprehensive methodology using electrophysiological recording and c-Fos staining is employed to demonstrate pulse repetition frequency (PRF)-dependent E/I selectivity of ultrasound stimulation in the top-down and bottom-up corticothalamic pathways of the visual circuit in rodents. Materials and methodsUltrasound stimulation at various PRFs is applied to either the lateral posterior nucleus of the thalamus (LP) or the primary visual cortex (V1), and multi-channel single-unit activity is recorded from the V1 using a silicon probe. Results and conclusionOur results demonstrate that high frequency PRFs, particularly at 3 kHz and 1 kHz, are effective at activating the bidirectional corticothalamic visual pathway. In addition, brain region-specific PRFs modulate E/I cortical signals, corticothalamic projections, and synaptic neurotransmission, which is imperative for circuit-specific applications and behavioral studies.

neuroscience↗

bioRxiv2025

Cis-regulatory Mutations Drive Tissue-specific Subfunctionalization of sRNA loci Regulating Soybean Seed Color During Domestication

Gene duplication and structural rearrangements have played a pivotal role in plant genome evolution, enabling functional divergence and the development of novel traits. In soybeans (Glycine max), the I locus, which regulates seed color through tissue-specific subfunctionalization of sRNA loci, offers an example of these mechanisms. Using long-read sequencing, we investigated structural variations underlying four major alleles (I, ii, ik, and i) at the I locus. Our study revealed that large-scale rearrangements, including duplications, inversions, and deletions, within a 180-kb CHS repeat-rich region drive allele-specific RNA silencing through small interfering RNAs (siRNAs). The dominant I allele contains a cis-regulatory region of DnaJ upstream of CHS genes, while the ii and ik alleles feature subtilisin- and P450-driven siRNA loci, respectively. The recessive i allele lacks siRNA production due to deletions or inversions disrupting CHS gene clusters. Phenotypic analyses and RNA-seq confirmed allele-specific, tissue-dependent expression of siRNAs correlating with seed coat pigmentation patterns. This study highlights the evolutionary role of repeat-rich regions in generating regulatory innovations and phenotypic diversity. Our findings underscore the importance of structural rearrangements in domestication traits and demonstrate the power of long-read sequencing for resolving complex genomic regions, advancing both evolutionary biology and crop improvement. SIGNIFICANCE{square} Naturally occurring mutations in the soybean seed coat have revealed the evolution of sRNA genes and their subfunctionalization, resulting in tissue-specific gene expression. {square}Non-homologous recombination of repetitive sequences drives cis-regulatory region rearrangements, leading to the subfunctionalization of sRNA loci that regulates one of domestication traits.

plant biology↗

bioRxiv2025

Cancer associated fibroblasts drive epithelial to mesenchymal transition and classical to basal change in pancreatic ductal adenocarcinoma cells with loss of IL-8 expression

Pancreatic ductal adenocarcinoma (PDAC) carries an extremely poor prognosis, in part resulting from cellular heterogeneity that supports overall tumorigenicity. Cancer associated fibroblasts (CAF) are key determinants of PDAC biology and response to systemic therapy. While CAF subtypes have been defined, the effects of patient-specific CAF heterogeneity and plasticity on tumor cell behavior remain unclear. Here, multi-omics was used to characterize the tumor microenvironment (TME) in tumors from patients undergoing curative-intent surgery for PDAC. In these same patients, matched tumor organoid and CAF lines were established to functionally validate the impact of CAFs on the tumor cells. CAFs were found to drive epithelial-mesenchymal transition (EMT) and a switch in tumor cell classificiaton from classical to basal subtype. Furthermore, we identified CAF-specific interleukin 8 (IL-8) as an important modulator of tumor cell subtype. Finally, we defined neighborhood relationships between tumor cell and T cell subsets. Statement of SignificanceThis multidimensional analysis highlights the diverse role CAFs have in influencing other cell types in the TME, including epithelial-derived tumor and infiltrating immune cells. Our methods provide a platform for evaluating emerging therapeutic approaches and for studying mechanisms that dictate tumor behavior in a manner that reflects patient-specific heterogeneity.

cancer biology↗

bioRxiv2024

Implicit modeling of the conformational landscape and sequence allows scoring and generation of stable proteins

Generative protein modeling provides advanced tools for designing diverse protein sequences and structures. However, accurately modeling the conformational landscape and designing sequences--ensuring that the designed sequence folds into the target structure as its most stable structure--remains a critical challenge. In this study, we present a systematic analysis of jointly optimizing P(structure|sequence) and P(sequence|structure), which enables us to find optimal solutions for modeling the conformational landscape. We support this approach with experimental evidence that joint optimization is superior for (1) designing stable proteins using a joint model (TrROS (TrRosetta) and TrMRF) (2) achieving high accuracy in stability prediction when jointly modeling (half-masked ESMFold pLDDT+ ESM2 Pseudo-likelihood). We further investigate features of sequences generated from the joint model and find that they exhibit higher frequencies of hydrophilic interactions, which may help maintain both secondary structure registry and pairing.

biophysics↗

bioRxiv2024

BindCraft: one-shot design of functional protein binders

Protein-protein interactions (PPIs) are at the core of all key biological processes. However, the complexity of the structural features that determine PPIs makes their design challenging. We present BindCraft, an open-source and automated pipeline for de novo protein binder design with experimental success rates of 10-100%. BindCraft leverages the weights of AlphaFold21 to generate binders with nanomolar affinity without the need for high-throughput screening or experimental optimization, even in the absence of known binding sites. We successfully designed binders against a diverse set of challenging targets, including cell-surface receptors, common allergens, de novo designed proteins, and multi-domain nucleases, such as CRISPR-Cas9. We showcase the functional and therapeutic potential of designed binders by reducing IgE binding to birch allergen in patient-derived samples, modulating Cas9 gene editing activity, and reducing the cytotoxicity of a foodborne bacterial enterotoxin. Lastly, we utilize cell surface receptor-specific binders to redirect AAV capsids for targeted gene delivery. This work represents a significant advancement towards a "one design-one binder" approach in computational design, with immense potential in therapeutics, diagnostics, and biotechnology.

bioinformatics↗

bioRxiv2024

Rho-ROCK liberates sequestered claudin for rapid de novo tight junction formation

The epithelial cell sheet maintains its integrity as a barrier while undergoing turnover of constituent cells. To sustain the barrier continuously, its essential to preserve the old tight junctions (TJs) between cells being excluded from the sheet and their neighbors while simultaneously forming de novo TJs between newly adjacent cells. However, the molecular mechanisms involved in the formation of de novo TJs remain largely unknown. This study investigates two scenarios: the formation of de novo TJs during the removal of apoptotic cells from monolayer epithelial sheets and during the differentiation of the granular layer in stratified epidermis. We revealed that rapid claudin assembly is achieved by actively regulating the dissociation of the EpCAM/TROP2-claudin complex in both situations. Furthermore, we found that the Rho-ROCK pathway initiates the activation of matriptase, which cleaves EpCAM/TROP2, resulting in the supply of polymerizable claudin from the stockpiled EpCAM/TROP2-claudin complex at the plasma membrane to induce rapid de novo TJ formation.

cell biology↗

bioRxiv2024

YAP inhibits NF-κB signaling and ccRCC growth by opposing p65-ZHX2 cooperativity

The prevailing view in the cancer field is that Hippo signaling pathway functions as a tumor suppressor pathway by blocking the oncogenic potential of the pathway effectors Yes1 associated transcriptional regulator (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ). However, YAP can also function as a context-dependent tumor suppressor in several types of cancer including clear cell renal cell carcinomas (ccRCC). We find that, in additional to inhibiting hypoxia-inducible factor 2 (HIF2), a major oncogenic driver in Von Hippel-Lindau (VHL)-/- ccRCC, YAP also blocks nuclear factor {kappa}B (NF-{kappa}B) signaling in ccRCC to inhibit cancer cell growth under conditions where HIF2 is dispensable. Mechanistically, YAP inhibits the expression of Zinc fingers and homeoboxes 2 (ZHX2), a VHL substrate and critical co-factor of NF-{kappa}B in ccRCC. Furthermore, YAP competes with ZHX2 for binding to the NF-{kappa}B subunit p65. Consequently, elevated nuclear YAP blocks the cooperativity between ZHX2 and the NF-{kappa}B subunit p65, leading to diminished NF-{kappa}B target gene expression. Pharmacological inhibition of Hippo kinase blocked NF-{kappa}B transcriptional program and suppressed ccRCC cancer cell growth, which can be rescued by overexpression of ZHX2 or p65. Our study uncovers a crosstalk between the Hippo and NF-{kappa}B/ZHX2 pathways and its involvement in ccRCC growth inhibition, suggesting that targeting the Hippo pathway may provide a therapeutical opportunity for ccRCC treatment.

cancer biology↗

bioRxiv2024

Differential growth enhancement followed by notable microbiota modulation in growing-finishing pigs by Bacillus subtilis strains ps4060, ps4100, and a 50:50 strain mixture

A 50:50 blend of two Bacillus subtilis strains positively impacted the productivity of finishing pigs. Given this observed effect, we hypothesized that each strain has distinct effects on weight gain and their influence on gut microbiota. In a 16-week test, 160 pigs were divided into four groups: basal diet, B. subtilis ps4100, B. subtilis ps4060, and 50:50 mixture supplemented. Subsequently, we compared body weight and fecal microbiota. Among the supplements, ps4100, ps4060, and the 50:50 mix yielded respective average daily weight gains (ADG) of 3.6%, 4.6%, and 3.9% by the 6th week. The weight difference was maintained through the 16th week. At the 11th week, the difference in -diversity among the fecal microbiota was marginal, and 17 of 229 genera showed differential abundance between the control and either of the treatment groups. A total of 12 of the 17 genera, including Lactobacillus, showed differential abundance between the ps4100 and ps4060-fed groups, and only Eubacterium consistently decreased in abundance in both the ps4100 and ps4060 groups. In comparison, microbial diversity was significantly different at the 16th week (p < 0.05), with 96 out of 229 genera exhibiting differential abundance. A total of 42 of the 96 genera exhibited similar patterns in both the ps4100 and ps4060 groups compared to the control group. Additionally, 236 of 687 microbial enzymes with differential abundance deduced from 16S rRNA reads showed similar differential abundance in both groups compared to the control group. We concluded that the overall microbial balance, rather than the dominance or significant decrease of a few specific genera, likely caused the enhanced ADG until the 11th week. Substantial changes in microbiota manifested at the 16th week did not cause dramatically increased ADG but were a consequence of weight gain and could positively affect animal physiology and health afterward.

microbiology↗

bioRxiv2024

Intrinsic skeletal muscle function and contraction-stimulated glucose uptake do not vary by time-of-day in mice

A growing body of data suggests that skeletal muscle contractile function and glucose metabolism vary by time-of-day, with chronobiological effects on intrinsic skeletal muscle properties being proposed as the underlying mediator. However, no studies have directly investigated intrinsic contractile function or glucose metabolism in skeletal muscle over a 24 h circadian cycle. To address this, we assessed intrinsic contractile function and endurance, as well as contraction-stimulated glucose uptake, in isolated extensor digitorum longus and soleus from female mice at four times-of-day (Zeitgeber Times 1, 7, 13, 19). Significantly, while both muscles demonstrated circadian-related changes in gene expression, intrinsic contractile function, endurance, and contraction-stimulated glucose uptake were not different between the four time points. Overall, these results demonstrate that time-of-day variation in exercise performance and the glycemia-reducing benefits of exercise are not due to chronobiological effects on intrinsic muscle function or contraction-stimulated glucose uptake. Impact statementEx vivo testing demonstrates that there is no time-of-day variation in the intrinsic contractile properties of skeletal muscle (including no effect on force production or endurance) or contraction-stimulated glucose uptake.

physiology↗

bioRxiv2024

Comparative specialization of intrinsic cardiac neurons in humans, mice, and pigs

Intrinsic cardiac neurons (ICNs) play a crucial role in the proper functioning of the heart; yet a paucity of data pertaining to human ICNs exists. We took a multidisciplinary approach to complete a detailed cellular comparison of the structure and function of ICNs from mice, pigs, and humans. Immunohistochemistry of whole and sectioned ganglia, transmission electron microscopy, intracellular microelectrode recording and dye filling for quantitative morphometry were used to define the neurophysiology, histochemistry, and ultrastructure of these cells across species. The densely packed, smaller ICNs of mouse lacked dendrites, formed axosomatic connections, and had high synaptic efficacy constituting an obligatory synapse. At Pig ICNs, a convergence of subthreshold cholinergic inputs onto extensive dendritic arbors supported greater summation and integration of synaptic input. Human ICNs were tonically firing, with synaptic stimulation evoking large suprathreshold excitatory postsynaptic potentials like mouse, and subthreshold potentials like pig. Ultrastructural examination of synaptic terminals revealed conserved architecture, yet small clear vesicles (SCVs) were larger in pigs and humans. The presence and localization of ganglionic neuropeptides was distinct, with abundant VIP observed in human but not pig or mouse ganglia, and little SP or CGRP in pig ganglia. Action potential waveforms were similar, but human ICNs had larger after-hyperpolarizations. Intrinsic excitability differed; 93% of human cells were tonic, all pig neurons were phasic, and both phasic and tonic phenotypes were observed in mouse. In combination, this publicly accessible, multimodal atlas of ICNs from mice, pigs, and humans identifies similarities and differences in the evolution of ICNs.

neuroscience↗

bioRxiv2024

Cancer-cell derived S100A11 promotes macrophage recruitment in ER+ breast cancer

Macrophages are pivotal in driving breast tumor development, progression, and resistance to treatment, particularly in estrogen receptor-positive (ER+) tumors, where they infiltrate the tumor microenvironment (TME) influenced by cancer cell-secreted factors. By analyzing single-cell RNA-sequencing data from 25 ER+ tumors, we elucidated interactions between cancer cells and macrophages, correlating macrophage density with epithelial cancer cell density. We identified that S100A11, a previously unexplored factor in macrophage-cancer crosstalk, predicts high macrophage density and poor outcomes in ER+ tumors. We found that recombinant S100A11 enhances macrophage infiltration and migration in a dose-dependent manner. Additionally, in 3D models, we showed that S100A11 expression levels in ER+ cancer cells predict macrophage infiltration patterns. Neutralizing S100A11 decreased macrophage recruitment, both in cancer cell lines and in a clinically relevant patient-derived organoid model, underscoring its role as a paracrine regulator of cancer-macrophage interactions in the pro-tumorigenic TME. This study offers novel insights into the interplay between macrophages and cancer cells in ER+ breast tumors, highlighting S100A11 as a potential therapeutic target to modulate the macrophage-rich tumor microenvironment.

systems biology↗

bioRxiv2024

Mapping the breast tumor microenvironment: proximity analysis reveals spatial relationships between macrophage subtypes and metastasis-initiating cancer cells

The development of metastasis, responsible for the majority of cancer-related fatalities, is the most dangerous aspect of breast cancer, the predominant malignancy affecting women. We previously identified specific cancer cell populations responsible for metastatic events which are cytokeratin-14 (CK14) and E-cadherin positive in luminal tumors, and E-cadherin and vimentin positive in triple-negative tumors. Since cancer cells evolve within a complex ecosystem comprised of immune cells and stromal cells, we sought to decipher the spatial interactions of these aggressive cancer cell populations within the tumor microenvironment (TME). We used imaging mass cytometry to detect 36 proteins in tumor microarrays containing paired primary and metastatic lesions from luminal or triple-negative breast cancers (TNBC), resulting in a dataset of 1,477,337 annotated cells. Focusing on metastasis-initiating cell populations, we observed close proximity to specific fibroblast and macrophage subtypes, a relationship maintained between primary and metastatic tumors. Notably, high CK14 in luminal cancer cells and high vimentin in TNBC cells correlated with close proximity to specific macrophage subtypes (CD163intCD206intPDL1intHLA-DR+ or PDL1highARG1high). Our in-depth spatial analysis elucidates that metastasis-initiating cancer cells exhibit with distinct cell populations within the TME, implicating the role of these cell-cell interactions in promoting metastasis.

cancer biology↗

bioRxiv2024

CAR T cell infiltration and cytotoxic killing within the core of 3D breast cancer spheroids under control of antigen sensing in microwell arrays

The success of chimeric antigen receptor (CAR) T cells in blood cancers has intensified efforts to develop CAR T therapies for solid cancers. In the solid tumor microenvironment, CAR T cell trafficking and suppression of cytotoxic killing represent limiting factors for therapeutic efficacy. Here, we present a microwell platform to study CAR T cell interactions with 3D tumor spheroids and determine predictors of anti-tumor CAR T cell function. To precisely control antigen sensing by CAR T cells, we utilized a switchable adaptor CAR system, that instead of directly binding to an antigen of interest, covalently attaches to co-administered antibody adaptors that mediate tumor antigen recognition. Following addition of an anti-HER2 adaptor antibody, primary human CAR T cells exhibited higher infiltration and clustering compared to the no adaptor control. By tracking CAR T cell killing at the individual spheroid level, we showed the suppressive effects of spheroid size and identified the initial CAR T cell : spheroid area ratio as a predictor of cytotoxicity. Spatiotemporal analysis revealed lower CAR T cell numbers and cytotoxicity in the spheroid core compared to the periphery. Finally, increasing CAR T cell seeding density, resulted in higher CAR T cell infiltration and cancer cell elimination in the spheroid core. Our findings provide new quantitative insights into CAR T cell-mediated killing of HER2+ breast tumor cells. Given the miniaturized nature and live imaging capabilities, our microfabricated system holds promise for discovering cell-cell interaction mechanisms that orchestrate antitumor CAR T cell functions and screening cellular immunotherapies in 3D tumor models.

bioengineering↗